Mass Spectrometric Methods for the Analysis of Heparin and Heparan Sulfate.

Glycosaminoglycans like heparin and heparan sulfate exhibit a high degree of structural microheterogeneity. This structural heterogeneity results from the biosynthetic process that produces these linear polysaccharides in cells and tissues. Heparin and heparan sulfate play critical roles in normal physiology and pathophysiology, hence it is important to understand how their structural features may influence overall activity. Therefore, high-resolution techniques like mass spectrometry represent a key part of the suite of methodologies available to probe the fine structural details of heparin and heparan sulfate. This chapter outlines the application of techniques like LC-MS and LC-MS/MS to study the composition of these polysaccharides, and techniques like GPC-MS that allow for an analysis of oligosaccharide fragments in these mixtures.

Circulating Markers of Inflammation Persist in Children and Adults With Giant Aneurysms After Kawasaki Disease.

BACKGROUND: The sequelae of Kawasaki disease (KD) vary widely with the greatest risk for future cardiovascular events among those who develop giant coronary artery aneurysms (CAA). We sought to define the molecular signature associated with different outcomes in pediatric and adult KD patients. METHODS: Molecular profiling was conducted using mass spectrometry-based shotgun proteomics, transcriptomics, and glycomics methods on 8 pediatric KD patients at the acute, subacute, and convalescent time points. Shotgun proteomics was performed on 9 KD adults with giant CAA and matched healthy controls. Plasma calprotectin was measured by ELISA in 28 pediatric KD patients 1 year post-KD, 70 adult KD patients, and 86 healthy adult volunteers. RESULTS: A characteristic molecular profile was seen in pediatric patients during the acute disease, which resolved at the subacute and convalescent periods in patients with no coronary artery sequelae but persisted in 2 patients who developed giant CAA. We, therefore, investigated persistence of inflammation in KD adults with giant CAA by shotgun proteomics that revealed a signature of active inflammation, immune regulation, and cell trafficking. Correlating results obtained using shotgun proteomics in the pediatric and adult KD cohorts identified elevated calprotectin levels in the plasma of patients with CAA. Investigation of expanded pediatric and adult KD cohorts revealed elevated levels of calprotectin in pediatric patients with giant CAA 1 year post-KD and in adult KD patients who developed giant CAA in childhood. CONCLUSIONS: Complex patterns of biomarkers of inflammation and cell trafficking can persist long after the acute phase of KD in patients with giant CAA. Elevated levels of plasma calprotectin months to decades after acute KD and infiltration of cells expressing S100A8 and A9 in vascular tissues suggest ongoing, subclinical inflammation. Calprotectin may serve as a biomarker to inform the management of KD patients following the acute illness.

Characterization of Endogenous Human FcγRIII by Mass Spectrometry Reveals Site, Allele and Sequence Specific Glycosylation.

The importance of IgG glycosylation, Fc-gamma receptor (FcγR) single nucleotide polymorphisms and FcγR copy number variations in fine tuning the immune response has been well established. There is a growing appreciation of the importance of glycosylation of FcγRs in modulating the FcγR-IgG interaction based on the association between the glycosylation of recombinant FcγRs and the kinetics and affinity of the FcγR-IgG interaction. Although glycosylation of recombinant FcγRs has been recently characterized, limited knowledge exists on the glycosylation of endogenous human FcγRs. In order to improve the structural understanding of FcγRs expressed on human cells we characterized the site specific glycosylation of native human FcγRIII from neutrophils of 50 healthy donors and from matched plasma for 43 of these individuals. Through this analysis we have confirmed site specific glycosylation patterns previously reported for soluble FcγRIII from a single donor, identified FcγRIIIb specific Asn45 glycosylation and an allelic effect on glycosylation at Asn162 of FcγRIIIb. Identification of FcγRIIIb specific glycosylation allows for assignment of FcγRIIIb alleles and relative copy number of the two alleles where DNA/RNA is not available. Intriguingly the types of structures found to be elevated at Asn162 in the NA2 allele have been shown to destabilize the Fc:FcγRIII interaction resulting in a faster dissociation rate. These differences in glycosylation may in part explain the differential activity reported for the two alleles which have similar in vitro affinity for IgG.

Demonstration of Biological and Immunological Equivalence of a Generic Glatiramer Acetate.

BACKGROUND: In April 2015, the US Food and Drug Administration approved the first generic glatiramer acetate, Glatopa® (M356), as fully substitutable for Copaxone® 20 mg/mL for relapsing forms of multiple sclerosis (MS). This approval was accomplished through an Abbreviated New Drug Application that demonstrated equivalence to Copaxone. METHOD: This article will provide an overview of the methods used to establish the biological and immunological equivalence of the two glatiramer acetate products, including methods evaluating antigenpresenting cell (APC) biology, T-cell biology, and other immunomodulatory effects. RESULTS: In vitro and in vivo experiments from multiple redundant orthogonal assays within four biological processes (aggregate biology, APC biology, T-cell biology, and B-cell biology) modulated by glatiramer acetate in MS established the biological and immunological equivalence of Glatopa and Copaxone and are described. The following were observed when comparing Glatopa and Copaxone in these experiments: equivalent delays in symptom onset and reductions in "disease" intensity in experimental autoimmune encephalomyelitis; equivalent dose-dependent increases in Glatopa- and Copaxone- induced monokine-induced interferon-gamma release from THP-1 cells; a shift to a T helper 2 phenotype resulting in the secretion of interleukin (IL)-4 and downregulation of IL-17 release; no differences in immunogenicity and the presence of equivalent "immunofingerprints" between both versions of glatiramer acetate; and no stimulation of histamine release with either glatiramer acetate in basophilic leukemia 2H3 cell lines. CONCLUSION: In summary, this comprehensive approach across different biological and immunological pathways modulated by glatiramer acetate consistently supported the biological and immunological equivalence of Glatopa and Copaxone.

An integrated approach using orthogonal analytical techniques to characterize heparan sulfate structure.

Heparan sulfate (HS), a glycosaminoglycan present on the surface of cells, has been postulated to have important roles in driving both normal and pathological physiologies. The chemical structure and sulfation pattern (domain structure) of HS is believed to determine its biological function, to vary across tissue types, and to be modified in the context of disease. Characterization of HS requires isolation and purification of cell surface HS as a complex mixture. This process may introduce additional chemical modification of the native residues. In this study, we describe an approach towards thorough characterization of bovine kidney heparan sulfate (BKHS) that utilizes a variety of orthogonal analytical techniques (e.g. NMR, IP-RPHPLC, LC-MS). These techniques are applied to characterize this mixture at various levels including composition, fragment level, and overall chain properties. The combination of these techniques in many instances provides orthogonal views into the fine structure of HS, and in other instances provides overlapping / confirmatory information from different perspectives. Specifically, this approach enables quantitative determination of natural and modified saccharide residues in the HS chains, and identifies unusual structures. Analysis of partially digested HS chains allows for a better understanding of the domain structures within this mixture, and yields specific insights into the non-reducing end and reducing end structures of the chains. This approach outlines a useful framework that can be applied to elucidate HS structure and thereby provides means to advance understanding of its biological role and potential involvement in disease progression. In addition, the techniques described here can be applied to characterization of heparin from different sources.

Adjuvanted influenza-H1N1 vaccination reveals lymphoid signatures of age-dependent early responses and of clinical adverse events.

Adjuvanted vaccines afford invaluable protection against disease, and the molecular and cellular changes they induce offer direct insight into human immunobiology. Here we show that within 24 h of receiving adjuvanted swine flu vaccine, healthy individuals made expansive, complex molecular and cellular responses that included overt lymphoid as well as myeloid contributions. Unexpectedly, this early response was subtly but significantly different in people older than ∼35 years. Wide-ranging adverse clinical events can seriously confound vaccine adoption, but whether there are immunological correlates of these is unknown. Here we identify a molecular signature of adverse events that was commonly associated with an existing B cell phenotype. Thus immunophenotypic variation among healthy humans may be manifest in complex pathophysiological responses.

Demonstration of equivalence of a generic glatiramer acetate (Glatopa™).

Glatiramer acetate (GA) has been available under the brand name Copaxone® for nearly two decades. Recently, the US Food and Drug Administration (FDA) approved the first generic GA, Glatopa™, as fully substitutable for all indications for which Copaxone 20mg is approved; Glatopa also represents the first FDA-approved "AP-rated," substitutable generic for treating patients with MS. Glatiramer acetate is a complex mixture of polypeptides and, consequently, its characterization presented challenges not generally encountered in drug development. Despite its complexity, and without requiring any clinical data, approval was accomplished through an Abbreviated New Drug Application in which equivalence to Copaxone was evaluated across four criteria: starting materials and basic chemistry; structural signatures for polymerization, depolymerization, and purification; physicochemical properties; and biological and immunological properties. This article describes the rigorous overall scientific approach used to successfully establish equivalence between Glatopa and Copaxone, and presents key representative data from several of the comprehensive sets of physicochemical (structural) and biological (functional) assays that were conducted.

Equivalent Gene Expression Profiles between Glatopa™ and Copaxone®.

Glatopa™ is a generic glatiramer acetate recently approved for the treatment of patients with relapsing forms of multiple sclerosis. Gene expression profiling was performed as a means to evaluate equivalence of Glatopa and Copaxone®. Microarray analysis containing 39,429 unique probes across the entire genome was performed in murine glatiramer acetate--responsive Th2-polarized T cells, a test system highly relevant to the biology of glatiramer acetate. A closely related but nonequivalent glatiramoid molecule was used as a control to establish assay sensitivity. Multiple probe-level (Student's t-test) and sample-level (principal component analysis, multidimensional scaling, and hierarchical clustering) statistical analyses were utilized to look for differences in gene expression induced by the test articles. The analyses were conducted across all genes measured, as well as across a subset of genes that were shown to be modulated by Copaxone. The following observations were made across multiple statistical analyses: the expression of numerous genes was significantly changed by treatment with Copaxone when compared against media-only control; gene expression profiles induced by Copaxone and Glatopa were not significantly different; and gene expression profiles induced by Copaxone and the nonequivalent glatiramoid were significantly different, underscoring the sensitivity of the test system and the multiple analysis methods. Comparative analysis was also performed on sets of transcripts relevant to T-cell biology and antigen presentation, among others that are known to be modulated by glatiramer acetate. No statistically significant differences were observed between Copaxone and Glatopa in the expression levels (magnitude and direction) of these glatiramer acetate-regulated genes. In conclusion, multiple methods consistently supported equivalent gene expression profiles between Copaxone and Glatopa.

A Quantitative Microtiter Assay for Sialylated Glycoform Analyses Using Lectin Complexes.

Fidelity of glycan structures is a key requirement for biotherapeutics, with carbohydrates playing an important role for therapeutic efficacy. Comprehensive glycan profiling techniques such as liquid chromatography (LC) and mass spectrometry (MS), while providing detailed description of glycan structures, require glycan cleavage, labeling, and paradigms to deconvolute the considerable data sets they generate. On the other hand, lectins as probes on microarrays have recently been used in orthogonal approaches for in situ glycoprofiling but require analyte labeling to take advantage of the capabilities of automated microarray readers and data analysis they afford. Herein, we describe a lectin-based microtiter assay (lectin-enzyme-linked immunosorbent assay [ELISA]) to quantify terminal glycan moieties, applicable to in vitro and in-cell glycan-engineered Fc proteins as well as intact IgGs from intravenous immunoglobulin (IVIG), a blood product containing pooled polyvalent IgG antibodies extracted from plasma from healthy human donors. We corroborate our findings with industry-standard LC-MS profiling. This "customizable" ELISA juxtaposes readouts from multiple lectins, focusing on a subset of glycoforms, and provides the ability to discern single- versus dual-arm glycosylation while defining levels of epitopes at sensitivities comparable to MS. Extendable to other biologics, this ELISA can be used stand-alone or complementary to MS for quantitative glycan analysis.

Controlled tetra-Fc sialylation of IVIg results in a drug candidate with consistent enhanced anti-inflammatory activity.

Despite the beneficial therapeutic effects of intravenous immunoglobulin (IVIg) in inflammatory diseases, consistent therapeutic efficacy and potency remain major limitations for patients and physicians using IVIg. These limitations have stimulated a desire to generate therapeutic alternatives that could leverage the broad mechanisms of action of IVIg while improving therapeutic consistency and potency. The identification of the important anti-inflammatory role of fragment crystallizable domain (Fc) sialylation has presented an opportunity to develop more potent Ig therapies. However, translating this concept to potent anti-inflammatory therapeutics has been hampered by the difficulty of generating suitable sialylated products for clinical use. Therefore, we set out to develop the first, to our knowledge, robust and scalable process for generating a well-qualified sialylated IVIg drug candidate with maximum Fc sialylation devoid of unwanted alterations to the IVIg mixture. Here, we describe a controlled enzymatic, scalable process to produce a tetra-Fc-sialylated (s4-IVIg) IVIg drug candidate and its qualification across a wide panel of analytic assays, including physicochemical, pharmacokinetic, biodistribution, and in vivo animal models of inflammation. Our in vivo characterization of this drug candidate revealed consistent, enhanced anti-inflammatory activity up to 10-fold higher than IVIg across different animal models. To our knowledge, this candidate represents the first s4-IVIg suitable for clinical use; it is also a valuable therapeutic alternative with more consistent and potent anti-inflammatory activity.

Mass spectrometric methods for the analysis of heparin and heparan sulfate.

Glycosaminoglycans like heparin and heparan sulfate exhibit a high degree of structural heterogeneity. This structural heterogeneity results from the biosynthetic process that produces these linear polysaccharides in cells and tissues. Heparin and heparan sulfate play critical roles in normal physiology and pathophysiology; hence it is important to understand how their structural features may influence overall activity. Therefore, high-resolution techniques like mass spectrometry represent a key part of the suite of methodologies available to probe the fine structural details of heparin and heparan sulfate. This chapter outlines the application of techniques like LC-MS and LC-MS/MS to study the composition of these polysaccharides, and techniques like GPC-MS that allow for an analysis of oligosaccharide fragments in these mixtures.

Synthesis and biological evaluation of a unique heparin mimetic hexasaccharide for structure-activity relationship studies.

To date, the structure-activity relationship studies of heparin/heparan sulfate with their diverse binding partners such as growth factors, cytokines, chemokines, and extracellular matrix proteins have been limited yet provide early insight that specific sequences contribute to this manifold biological role. This has led to an impetus for the chemical synthesis of oligosaccharide fragments of these complex polysaccharides, which can provide an effective tool for this goal. The synthesis of three heparin mimetic hexasaccharides with distinct structural patterns is described herein, and the influence of the targeted substitution on their bioactivity profiles is studied using in vitro affinity and/or inhibition toward different growth factors and proteins. Additionally, the particularly challenging synthesis of an irregular hexasaccharide is reported, which, interestingly, in spite of being considerably structurally similar with its two counterparts, displayed a unique and remarkably distinct profile in the test assays.

Characterization of heparin-protein interaction by saturation transfer difference (STD) NMR.

The binding affinity and specificity of heparin to proteins is widely recognized to be sulfation-pattern dependent. However, for the majority of heparin-binding proteins (HBPs), it still remains unclear what moieties are involved in the specific binding interaction. Here, we report our study using saturation transfer difference (STD) nuclear magnetic resonance (NMR) to map out the interactions of synthetic heparin oligosaccharides with HBPs, such as basic fibroblast growth factor (FGF2) and fibroblast growth factor 10 (FGF10), to provide insight into the critical epitopes of heparin ligands involved. The irradiation frequency of STD NMR was carefully chosen to excite the methylene protons so that enhanced sensitivity was obtained for the heparin-protein complex. We believe this approach opens up additional application avenues to further investigate heparin-protein interactions.

Structural elucidation of the tetrasaccharide pool in enoxaparin sodium.

Low-molecular-weight heparins (LMWHs) are produced from heparin by various depolymerization strategies, which result in a reduction of the average molecular weight of the polysaccharide chains, a reduction of the anti-factor IIa activity (and a concomitant increase in the anti-factor Xa/anti-factor IIa ratio), and introduction of process-related structural signatures. Numerous techniques have been developed to characterize LMWHs and to measure the type and extent of structural modifications that are introduced as a function of the depolymerization process. We present here an analysis of the tetrasaccharide pool of enoxaparin sodium, a LMWH produced by chemical ß-elimination of heparin benzyl ester. We identify the predominant sequences present within the tetrasaccharide pool and demonstrate that this pool provides a sensitive, specific readout of the physicochemical process conditions used to generate enoxaparin sodium.

Identification of a novel structure in heparin generated by sequential oxidative-reductive treatment.

Unfractionated heparin is isolated from animal organs, predominantly porcine intestinal mucosa, and goes through an extensive process of purification before it can be used for pharmaceutical purposes. While the structural microheterogeneity of heparin is predominantly biosynthetically imprinted in the Golgi, subsequent steps involved in the purification and manufacture of commercial heparin can lead to the introduction of additional modifications. Postheparin crisis of 2008, it has become increasingly important to identify what additional structural diversity is introduced as a function of the purification process and thus can be determined as being heparin-related, as opposed to being an adulterant or contaminant, e.g., oversulfated chondroitin sulfate. Our study focuses on the identification of a previously unreported structure in heparin that arises due to specific steps used in the manufacturing process. This structure was initially observed as a disaccharide peak in a complete enzymatic digest of heparin, but its presence was later identified in the NMR spectra of intact heparin as well. Structural elucidation experiments involved isolation of this structure and analysis based on multidimensional NMR and liquid chromatography coupled with mass spectrometry (LC-MS). Heparin was also subjected to specific chemical reactions to determine which steps in the manufacturing process are responsible for this novel structure. Our results allowed for the definitive assignment of the structure of this novel process-related modification and enabled an identification of the putative steps in the process that give rise to the structure.